Structure and regulation of DNA condensates by disordered linker histone tails

对控制基因转录活性的基本因素的研究是所有生物体的基础生物学的基础，只有当浓缩的基因组DNA（染色质）的物理性质得到很好的描述时，才能正确理解这些过程。考虑到这一点，也许令人惊讶的是，还有这么多东西需要理解。两米长的DNA浓缩在每个细胞核中。这分两个阶段实现。第一阶段包括将DNA缠绕在蛋白质线轴上，形成类似于绳子上的珠子的“核小体”。第二个阶段是将这种结构进一步压缩成一种更紧凑的结构;这一阶段还不太清楚。生物学可以从自上而下（即观察细胞）或自下而上（即观察原子水平的分子）进行研究;人们总是希望它们结合起来形成一幅一致的图景，从而克服固有的局限性，并验证每种方法产生的模型。这两种方法都是必要的，以充分了解大多数生物过程。在染色质的情况下，目前有一个不连续的关于第二阶段的凝聚，因为30纳米的纤维预测形成的第二阶段由自下而上的方法还没有观察到的最好的新成像技术应用于活细胞。相反，纤维看起来更开放，灵活，无序和异质，自组装成具有液体性质的大染色质球。高度有序的30 nm纤维似乎可能代表了染色质浓缩和失活的极端情况，并且与转录相关的情况可能更具动态性。这种观点与我们和其他人从不同的自下而上方法中出现的一些替代观点产生了共鸣，这些观点发现了包装DNA的蛋白质中的高水平内在无序，以及它们将DNA浓缩成致密液体凝聚物的能力，其中保留了纤维的动力学。液体凝聚物是一种令人信服的和更合理的手段，通过它染色质可以快速响应环境刺激。我们已经开发了一个模型系统，使我们能够研究，在原子水平的分辨率，DNA包装蛋白质的方式-特别是“连接组蛋白”-结合到DNA，以及蛋白质/DNA复合物相分离成致密液滴的条件。它还允许热力学测量。我们想利用这个系统来回答几个关键问题：DNA到底是如何被连接组蛋白结合和浓缩的？所得到的冷凝物的高度拥挤的环境是否允许已知在体内起作用的修饰酶进入并起作用，冷凝物如何反应？蛋白质/DNA复合物的内在紊乱如何促进与其他染色质相关蛋白质的快速交换？在冷凝物中存在什么更高级的结构，如果有的话，它们的装配是如何控制和调节的？许多连接组蛋白亚型（例如，人类中有11种微妙不同的连接组蛋白，它们要么在细胞的生命周期中出现，要么在精子或卵子中定位）和DNA序列在内容和修饰上不同，例如那些标记基因开始的序列或那些通过添加甲基而“沉默”的序列之间的基因组凝聚的机制相似性/差异是什么？总而言之，基因组的浓缩是一个基本过程，它通过各种机制在生命王国中发生。总的来说，真核生物分两个阶段实现。第一种是通过核小体的形成，这已经得到了大量的研究和很好的理解。第二个是它通过连接体组蛋白的进一步浓缩，这一点还不太清楚，本提案将解决这一问题。

Study of the fundamental factors controlling the transcriptional activity of genes underpins the basic biology of all organisms, and the processes can only be properly understood if the physical nature of condensed genomic DNA (chromatin) is well described. Given this, it is perhaps surprising that there is so much left to understand. Two meters of DNA are condensed into each cell nucleus. This is achieved in two stages. The first stage involves wrapping the DNA around protein spools to form "nucleosomes" that resemble beads on a string. The second stage involves the further condensation of this structure into one that is more compact; this stage is less well understood.Biology can be approached from the top down (i.e. looking at cells) or bottom up (i.e. looking at atomic-level molecules); the hope is always that they join up to make a consistent picture, thus overcoming the inherent limitations and validating the model generated from each approach. Both approaches are necessary to fully understand most biological processes.In the case of chromatin, there is currently a discontinuity concerning the second stage of condensation, since the 30 nm fibre predicted to form in the second stage by a bottom-up approach has not been observed by the best new imaging techniques applied to live cells. Instead, the fibre appears more open, flexible, disordered and heterogeneous, self-assembling into large chromatin globules with liquid-like properties. It seems likely that the highly ordered 30 nm fibre may represent an extreme case of condensed and inactive chromatin, and the more transcriptionally-relevant situation may be much more dynamic. This view is resonant with some alternative views that are emerging from different bottom-up approaches by us and others, that pick up on the high level of inherent disorder in the proteins that package DNA, and their ability to concentrate the DNA into dense liquid condensates, in which the dynamics of the fibre are retained. A liquid condensate is a compelling and more plausible means by which chromatin could respond quickly to environmental stimuli.We have developed a model system that allows us to study, at atomic-level resolution, the way DNA-packaging proteins - specifically the 'linker histones' - bind to the DNA, and the conditions under which the protein/DNA complexes phase separate into dense liquid droplets. It also permits thermodynamic measurements. We would like to exploit this system to answer several key questions: exactly how is DNA bound and condensed by linker histones?; does the highly crowded environment of the resulting condensate allow the entry and action of modifying enzymes known to act in vivo, and how do the condensates respond?; how does the intrinsic disorder of the protein/DNA complexes facilitate rapid exchange by and with other chromatin-associated proteins?; what higher-order structures are present in the condensates, if any, and how is their assembly controlled and regulated?; what are the mechanistic similarities/differences in genome condensation between the many linker histone subtypes (there are for example 11 subtly different linker histones in humans that either come and go through the lifetime of a cell, or locate to sperm or egg) and DNA sequences differing in content and modifications, such as those marking the start of genes or those that are 'silenced' by the addition of methyl groups?In summary, the condensation of the genome is a fundamental process that occurs by a variety of mechanisms across the kingdoms of life. By and large, eukaryotes achieve it in two stages. The first is through the formation of nucleosomes, which has been heavily studied and is well understood. The second is its further condensation by linker histones, which is less well understood, and will be addressed by this proposal.